Human pluripotent stem cells (hPSCs), including human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs), can proliferate indefinitely in culture while maintaining the capability to differentiate into multiple types of somatic cells. These cells are greatly valued as providing unlimited cell source in cell therapy and regenerative medicine. As demonstrated by recent FDA approval of clinical trials, human embryonic stem cell (hESC)-based cell therapies are progressing from bench to clinic. However, currently available plate and T-flask-based culture platforms severely limit the scalability of hPSCs production to commercially relevant lot sizes. To unleash the potential of hPSCs in cell therapy and regenerative medicine, a scalable hPSC manufacturing process must be developed. Scaling up existing flask-based processes is a critical stepping stone in translating current hPSC research into clinical application. One of the biggest challenges is to establish a scalable passaging method for large scale multilayer vessels that maintains high yield, pluripotent phenotype, and karyotypic stability.
Traditionally, hPSCs are harvested and passaged as colony fragments by mechanical scraping with or without pre-treatment with enzymes (such as collagenase or Dispase®). This process is labor intensive and cannot be applied in culturing hPSCs in multilayer cell culture vessels, the platform widely used in producing commercial scale adherent cells. Cells growing in multilayer cell culturing vessels cannot be accessed for scraping. In addition, mechanical scraping causes damage to cells. Without scraping, cell viability can increase up to 90 percent. Known methods related to single-cell passaging and harvesting are not desirable due for example, to concerns related to low post-passaging and cryopreservation recovery and abnormal karyotype, associated with low cloning efficiency of hPSCs. Although Rho-associated kinase inhibitors (ROCK Inhibitors) have been reported to be able to improve hPSC cloning efficiency, the mechanism is not fully understood and the effect of ROCK Inhibitors on hPSC culture is yet to be evaluated. Therefore, passaging hPSCs as single cells in the presence of ROCK Inhibitors is not widely accepted.
Recently, passaging hESCs with non-enzymatic cell detachment solutions, mainly EDTA (ethylene diamine tetraacetic acid) solutions, has been adopted by some hESC labs and is spreading from academic labs into industry. One of the commercially available EDTA-containing solutions for cell dissociation is Versene® EDTA, which contains 0.55 mM EDTA and has been used for harvesting and passaging hPSCs. The typical procedure of passaging hESCs with Versene® EDTA starts with washing the culture with Ca2+/Mg2+-free buffer (for example, Dulbecco's phosphate-buffered saline; DPBS), followed by incubating the culture in Versene® EDTA for 4-9 minutes. Versene® EDTA is then removed and cells are physically removed from the surface as clusters by manual hosing of the cells with culture medium via pipetting. Compared with the conventional enzymatic-treatment-followed-by-scraping method (see Table 1), the advantage of this method is that (1) it uses a non-enzymatic solution—thus, there is no need for post-detachment washing or centrifugation to eliminate enzyme, and (2) it does not require scraping—the cells treated with Versene® EDTA can be washed off the surface. As described in Table 1, and illustrated in FIG. 1, compared with hESCs treated with enzyme and scraped off the culturing surface, the hESCs treated with Versene® EDTA and detached without scraping have higher post-detachment viability and re-attach to the new culturing surface much faster (minutes vs. hours) when passaged.
TABLE 1Methods of Harvesting/Passaging hESCsConventional Enzymatic and Scraping MethodVersene ® EDTA Method1. remove culture medium1. remove culture medium2. incubate in collagenase or Dispase ® at 37° C.2. wash once with Ca2+/Mg2+-freefor 2-5 minutesbuffer (for example, DPBS)3. remove collagenase or Dispase ®3. incubate in Versene ® EDTA atroom temperature for 4-9 minutes4. wash three times with culture medium4. remove Versene ® EDTA5. scrape hESCs off the surface in culture medium5. hose the cells off the surface withwith cell scraper or pipette tipculture medium6. collect the colony clumps (harvest) or transfer6. collect the cell clusters (harvest) orinto a fresh culture vessel (passage)transfer into fresh culture vessel(passage)
However, when applied into expanding hESC in multilayer vessels, the Versene® EDTA passaging/harvesting method is not ideal. Versene® EDTA seems to breaks down cell-cell association faster than it breaks cell-surface bonding. If hESC culture is over-treated with Versene® EDTA (>9 minutes), a greater percentage of cells come off the surface as single cells rather than clusters or clumps. In order to avoid getting too many single cells, the treatment time normally is controlled between seven to nine minutes. After the removal of Versene® EDTA, in six-well plate or T-flask culture format, fluidic shear force generated by hosing with culture medium via manual pipetting is needed to dislodge the cells off the surface. However, hESC culture in multilayer vessels cannot be manually sheared with culture medium as pipettes cannot be introduced inside the vessels. Instead, in this culture format, after Versene® EDTA is replaced with culture medium, vigorous tapping is applied to dislodge the cells. The mechanical force (tapping) has to follow the replacement of Versene® EDTA with culture medium immediately because Versene® EDTA treated hESCs quickly re-attach to the surface once they come in contact with culture medium. In fact, with the current state-of-art, it is only possible to harvest 40-70% of the entire culture in multilayer cell factories—dramatically impacting the yield of these very expensive cells. One possible solution to increase the yield is not to replace Versene® EDTA with culture medium and to dislodge the cells in Versene® EDTA instead. However, in this case, the exposure time of cells to Versene® EDTA is increased, which increases the risk of getting too many single cells and obtaining karyotypic unstable colonies. In addition, extra steps of post-detachment processing have to follow to remove or neutralize Versene® EDTA from the final harvest, which will not only add to the labor intensity but also further the breakup of the small hESC clusters.
There is therefore a need for a scalable and high-yielding passaging and harvesting formulation and method for hPSCs that eliminates or reduces the drawbacks of methods known in the art.